Isaac Cao, Theoneste Hakorimana and Kurtis Nokes PARE Project Isaac Cao, Theoneste Hakorimana and Kurtis Nokes 2018 Fall Semester Kergerye

Introduction The PARE project is a citizen-science research project that records and monitor antibiotic resistances organism. In other words, we are trying to find organisms that are resistance to antibiotic in our soil. From doing this research project, we are able to provide an in-depth contribution to not only for scientific community, but as well as in the health community.

Soil Sample Collection Data The soil we collected was at a park that have frequent visitors that comes and goes to relax and have fun. Since, this area is visited so much, there are bound to have people who play in the dirt and bring unwanted guest at home or anywhere else. Fassio Farm Park, 3720 So. 5200 W, WVC, UT 84120 Latitude: 40.6915 & Longitude: -112.0161 Town park/Playground/Dog park Collection Date: 10/25/18 @ 1130 with Cloudy weather condition

PARE Data Results Colony collected Colony Collected 5 day period before plated Culture medium: lMAC Incubation temp: 28 C Hours of incubated: 72 hours Used Tet3 (10^3) for set 1 and 2 for Agarose Gel Electrophoresis

PARE Data result continue Percent tetracycline resistant cells CFUs Per gram soil Plate set 1 Plate Set 2 Plate 1 Plate 2 # Colonies 216 68 Volume of Cell plated 0.2 mL Dilution Factor 10^4 10^5 Total CFU/gram soil 1.08x10^7 3.4x10^7 Tetracycline concentration 3 ug/ml 30ug/ml 30 ug/ml # Colonies 210 58 227 38 Volume 0.2 mL Dilution Factor 10^3 10 Total Tet CFU/gram soil 1.05x10^6 2.9X10^3 1.135x10^6 1.9x10^3 Percent of Tet CFU 9.72% 0.027% 3.36% 0.0056% Table 1 Table 2

Agarose Gel Electrophoresis Results Results are on the Second row Well 2 (17433) for set 1 ~750 BP Well 3 (17434) for set 2 <50 BP Both got analyzed

DNA Sequencing Results Set 1: 17433 1000 in length, ~750 Base Pairs **NNNNNTCNANNGNGGTACCGTCCTCCCGAAGGTTAGACTAAGCTACTTCTGGTGCAA CCCACTCCCATGGTGTGACGGGCGGTGTGTACAAGGCCCGGGAACGTATTCACCGCGAC ATTCTGATTCGCGATTACTAGCGATTCCGACTTCACGCAGTCGAGTTGCAGACTGCGATC CGGACTACGATCGGTTTTATGGGATTAGCTCCACCTCGCGGCTTGGCAACCCTTTGTACC GACCATTGTAGCACGTGTGTAGCCCAGGCCGTAAGGGCCATGATGACTTGACGTCATCC CCACCTTCCTCCGGTTTGTCACCGGCAGTCTCCTTAGAGTGCCCACCATAACGTGCTGGTA ACTAAGGACAAGGGTTGCGCTCGTTACGGGACTTAACCCAACATCTCACGACACGAGCT GACGACAGCCATGCAGCACCTGTCTCAATGTTCCCGAAGGCACCAATCCATCTCTGGAAA GTTCATTGGATGTCAAGGCCTGGTAAGGTTCTTCGCGTTGCTTCGAATTAAACCACATGC TCCACCGCTTGTGCGGGCCCCCGTCAATTCATTTGAGTTTTAACCTTGCGGCCGTACTCCC CAGGCGGTCAACTTAATGCGTTAGCTGCGCCACTAAGAGCTCAAGGCTCCCAACGGCTA GTTGACATCGTTTACGGCGTGGACTACCANGGTATCTAATCCTGTTTGCTCCCCACGCTTT CGCACCTCANTGTCAGTATCAGTCCNGTGGTCGCCTTCGCCACTGGTGTTCCTTCCTATAT CTACGCATTTCACCGCTACACANGAAATTCCACCACCCTCTACCANACTCTAGCTTGCCAG TTTTGGATGCANTTCCCAGGTTGAGCCCCGGGATTTNNCATCCCACTTAANNAAANNNC CTACGCGCGCTNACGCCAGTAATTCCNANNAANGCNTGCACCCCNCTGNANTACCGCN NNGCNGGCNNNNNNNNNNNNNNGCTNATTCTGTNGNANNTNNNNN** Set 2: 17434 58 in length, Less than 50 Base Pairs Not enough base pair to run the BLAST program. Most likely cause of the small sequences can be that we did not put in enough of our colony in the PCR solution for Set 2. **NNNNNNANNTNNNNNNNNNNNNNNNNNTNATCTGAGCCATCGNTCTAA CNNNNNNNCT**

BLAST Results and Identification Uncultured Bacterium gene for 16S rRNA partial sequence, clone: HSM050P-B-4 Query Cover: 94%, E value: 0, Ident: 97% PCR-derived sequence from peat soil (Turf); accumulation of partially decayed vegetation or organic matter. Masaru Akiyama Horonobe Research Institute for the Subsurface Environment (H-RISE), Northern Advancement Center for Science & Technology; Sakae-machi 5-3, Horonobe, Hokkaido 098-3221, Japan

Phylogenetic Tree Specimen that are closely match for BLAST results

Pseudomonas sp. 55A1 Pseudomonas sp. 55A1 16S ribosomal RNA gene, partial sequence -Second Match on BLAST w/ a name of an organism Query Cover: 94%, E value: 0, Ident: 97% Proteobacteria; Gammaproteobacteria; Pseudomonadales; Pseudomonadaceae; Pseudomonas. Becker,M.H., Walke,J.B., Murrill,L., Woodhams,D.C., Reinert,L.K., Rollins-Smith,L.A., Burzynski,E.A., Umile,T.P., Minbiole,K.P. and Belden,L.K.

Summary From obtaining soil to analyzing it genetic code we were able to learn so much regarding our project. We were able to decoded one of our colony, and learn that this organism is resistant to a low dose of the tetracycline antibiotic. In addition, as we increase the dose of the tetracycline, we see a decrease trend in colonies being formed. So, if we have kept going with increasing the dose on our plate then we would have seen fewer and fewer growths in the plates. Even though we were able to decode one of our colony we were able to learn so much about it on the BLAST search. From the people who discover the organism to the family tree of that organism. From all this we were able to learn that even a local park can contain antibiotic resistant organism. So, let your biology department, and even your local high school to get involved in the PARE project. If we increase our knowledge of antibiotic resistant organisms we may benefit not only as a community but as a whole nation. As knowledge is power and with that power we can spread and teach others on how to be safe against these organisms. In addition, provide more resources so that future scientist we could try this experiment and hopefully use different antibiotics than the tetracycline to see if any other organisms are resistant. From this we can improve the PARE database and the near future could be uses for future experiments with antibiotic resistance organisms. In conclusion, this research project was able to provide an in-depth contribution to not only for scientific community, but as well as in the health community. As we hope that this information will teach you how to be safe against these organisms.

Interesting Website regarding the Organism -Antibiotic resistance in Pseudomonas Research- https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4265834/ -Sign and Symptoms of Pseudomonas Infections- https://www.cdc.gov/hai/organisms/pseudomonas.html -Antibiotic resistance Research on Gammaproteobacteria https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4356103/ -Increase trend of Antibiotic Resistant Organism https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4378521/